Heterocarpine, a human ghrh-binding protein

ABSTRACT

The invention relates to a human GHRH-binding protein (human Growth Hormone-releasing hormone). Said protein, which is obtained from the Pilocarpus Heterophyllus plant, can be used to prepare medicaments intended to antagonise the effects of GHRH and to treat proliferative diseases (particularly cancer), acromegaly or diabetic nephropathies and retinopathies.

[0001] The present invention relates to a human GHRH-binding (humanGrowth Hormone releasing hormone) protein.

[0002] Growth hormone (“GH”) is a protein with 191 amino acids whichstimulates the production of a number of growth factors, such asInsulin-Like Growth Factor I (IGF-1) and triggers the growth of a largenumber of tissues (skeleton, connective tissues, muscles and viscera).GH also has physiological activities, increasing the synthesis of thenucleic acids, proteins and lipolysis whilst reducing urinary secretions(Frohman L. A. & Kineman, R. D., Handbook of Physiology, HormonalControl of Growth, edited by Kostyo, J. L. & Goodman, H. M. (OxfordUniv. Press, New York. , 1999), p. 189-221).

[0003] The synthesis of GH is regulated by factors with positive ornegative action secreted by the hypothalamus. The main factorcontrolling the production of GH is the “Growth Hormone ReleasingHormone”(GHRH), a peptide with 44 amino acids in humans.

[0004] GH and GHRH are involved in a number of diseases. Of these, thefollowing should be mentioned in particular: cancer (in particularprostate or lung cancer), acromegaly, diabetic retinopathies andnephropathies; for these pathologies, treatment with GHRH antagonists isindicated. Due to the number of diseases potentially concerned, theindustry continues to research GHRH antagonists.

[0005] The Applicant has therefore just isolated a new protein ofvegetable origin, which has the property of binding human GHRH.

[0006] A first subject of the invention is therefore an isolated proteinwhich can be obtained by extraction from the plant Pilocarpusheterophyllus, which is characterized in that it has a molecular mass ofapproximately 90.9 kDa and comprises fragments of peptide sequencesSEQ.ID.NO. 1, SEQ.ID.NO. 2 and SEQ.ID.NO. 3, said protein being able tobe presented in a glycosylated or non-glycosylated form. In order tosimplify the disclosure which follows, this protein is hereafter called“heterocarpine”.

[0007] Said SEQ.ID.NO. 1, SEQ.ID.NO. 2 and SEQ.ID.NO. 3 sequences are asfollows: SEQ.ID.NO.1: KLIGARYFDK SEQ.ID.NO.2: YGEDIIVGVLDSGVSEQ.ID.NO.3: PESESY

[0008] The nomenclature used above (as in the remainder of the presentApplication) in order to define the peptides is that specified by the“IUPAC-IUB Commission on Biochemical Nomenclature” in which, inaccordance with the standard representation, the N-terminal amino acid(amino group) appears on the left and the C-terminal amino acid(carboxyl group) appears on the right. The term “natural amino acid”indicates one of the natural L-amino acids found in the naturalproteins: Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu,Gln, Cys, Met, Phe, Tyr, Pro, Trp and His.

[0009] A protein is called “isolated” if it is taken out of its originalenvironment. In particular, a natural protein is isolated if it isseparated from the biological material with which it coexists in thenatural system.

[0010] The invention preferably relates to heterocarpine in itsnon-glycosylated form.

[0011] According to a preferred variant of the invention, theheterocarpine is obtained from an extract of cells of the plantPilocarpus Heterophyllus cultured in vitro.

[0012] Moreover, a subject of the invention is also a monoclonalantibody, or an antigen binding fragment of the latter, whichspecifically binds heterocarpine.

[0013] Heterocarpine has the property of binding human GHRH. In vitro,heterocarpine binds human GHRH and thus inhibits the synthesis of cyclicAMP induced during the binding of human GHRH on its receptor. In vivo,in rats, the heterocarpinehuman GHRH complex is formed in the bloodcompartment and, in dose-dependent manner inhibits the GH synthesisinduced by 10 μg of human GHRH in a mole to mole ratio. Theheterocarpine has the property of binding the human GHRH.

[0014] These properties render the compounds of the invention suitablefor a pharmaceutical use. Therefore, a subject of the invention is also,as a medicament, heterocarpine in glycosylated or non-glycosylated form.It also relates to pharmaceutical compositions containing, as activeingredient, heterocarpine in a glycosylated or non-glycosylated form,said composition also comprising one or more pharmaceutically acceptableexcipients. A further subject is the use of heterocarpine in aglycosylated or nonglycosylated form for preparing medicaments intendedto antagonize the effects of GHRH, to treat proliferative diseases (andin particular cancer), to treat acromegaly or to treat diabeticretinopathies and nephropathies. With regard to cancer, heterocarpine isparticularly suitable for preparing a medicament intended to treatcarcinoid and pancreatic tumours, hypothalamo-hypophysealgangliocytomas, bronchial, intestinal and hepatic carcinomas,sympathoadrenergic tumours, pheochromocytomas, hypophyseal adenomas andthyroid carcinomas.

[0015] A subject of the invention is also, as a medicament, a monoclonalantibody, or an antigen binding fragment of the latter, whichspecifically binds heterocarpine. It also relates to a pharmaceuticalcomposition comprising, as active ingredient, a monoclonal antibody, oran antigen binding fragment of the latter, which specifically bindsheterocarpine, said composition also comprising one or morepharmaceutically acceptable excipients. It relates moreover to the useof a monoclonal antibody, or of an antigen binding fragment of thelatter, which specifically binds heterocarpine, for preparingmedicaments intended to antagonize the effects of GHRH, to treatproliferative diseases (and in particular cancer), to treat acromegalyor to treat diabetic retinopathies and nephropathies. As regards cancer,said monoclonal antibody or said antigen binding fragment of the latteris particularly suitable for preparing a medicament intended to treatcarcinoid and pancreatic tumours, hypothalamo-hypophysealgangliocytomas, bronchial, intestinal and hepatic carcinomas,sympathoadrenergic tumours, pheochromocytomas, hypophyseal adenomas andthyroid carcinomas.

[0016] The invention also relates to the use of heterocarpine as anexcipient in a pharmaceutical composition intended for the controlledrelease of GHRH. It also relates to a pharmaceutical compositioncomprising GHRH, heterocarpine and one or more pharmaceuticallyacceptable excipients.

[0017] Finally, other subjects of the invention are the processes makingit possible to extract and isolate heterocarpine from cells of the plantPilocarpus Heterophyllus, said cells preferably originating from invitro cultures. These processes essentially include a stage ofextraction of the cells from the plant Pilocarpus Heterophylluswithwater at a temperature of 0 to 50° C., and preferably 4 to 25° C., saidextraction stage being followed by a filtration stage to separate theheterocarpine-rich filtrate from the Pilocarpus Heterophyllus cells andby one or more stages of separation of the heterocarpine from the othercomponents extracted from the plant Pilocarpus Heterophyllus.

[0018] According to a first variant, these extraction and isolationprocesses essentially include the following successive stages:

[0019] a) a stage of extraction of the cells from the plant PilocarpusHeterophyllus with water at a temperature of 0 to 50° C., and preferablyof 4 to 25° C., said extraction stage being followed by a filtrationstage to separate the heterocarpine-rich filtrate from the PilocarpusHeterophyllus cells;

[0020] b) a stage of precipitation of the proteins extracted, forexample by adding ammonium sulphate, followed by a stage of separationof the precipitate (by filtration or, preferably, by centrifugation);

[0021] c) solubilizing the precipitates recovered in Stage b) in water;and

[0022] d) a stage of gel-filtration chromatography in order to separatethe heterocarpine from the other components of the solution.

[0023] According to another variant, these extraction and isolationprocesses essentially include the following successive stages:

[0024] a) a stage of extraction of the cells from the plant PilocarpusHeterophyllus with water at a temperature of 0 to 50° C., and preferably4 to 25° C., said extraction stage being followed by a filtration stageto separate the heterocarpine-rich filtrate from the PilocarpusHeterophyllus cells;

[0025] b) a stage of delipidation of the solution obtained in a),acidified by the addition of a non-oxidizing acid (for examplehydrochloric acid, sulphuric acid or phosphoric acid) at a pH preferablycomprised between 2 and 4, using liquid-liquid extraction (preferably byusing an organic solvent such as dichloromethane, heptane, hexane orcyclohexane);

[0026] c) a stage of elimination of the tannins by bringing thedelipidated solution obtained in c) into contact withpolyvinylpyrrolidone (or also nylon 66) followed by filtration onlarge-pore resin (preferably a polystyrene-based resin such as the resinDiaion® HP20);

[0027] d) adjusting the filtrate obtained after Stage c) to alkaline pH(preferably between pH 9 and 11) by the addition of a base such asammonium hydroxide, sodium hydroxide or potassium hydroxide;

[0028] e) one or more stages of filtration on anion-exchange resin, theeluent for this stage or these filtration stages preferably being abuffer solution having a pH between 9 and 11 and optionally containingconcentration gradients of a salt (such as for example sodium chlorideor ammonium sulphate), in order to separate the heterocarpine from theother components of the solution; and

[0029] f) a desalination stage consisting of passing the solutionobtained in Stage e) over a resin separating the constituents of amixture on the basis of their molecular mass (such as the resinSephadex® G25 or Superdexe® 200 HR) and the elution of this mixture oversaid resin with water.

[0030] The pharmaceutical compositions containing a compound of theinvention can be in solid form such as, for example, powders, pills,granules, tablets, liposomes, gelatin capsules or suppositories. Thepills, tablets or gelatin capsules can be coated with a substancecapable of protecting the composition from the action of gastric acid orthe enzymes in the subject's stomach for a sufficient period of time toallow this composition to pass undigested into the latter's smallintestine. The compound can also be administered locally, for example tothe actual site of a tumour. The compound can also be administeredaccording to a sustained-release process (for example by using asustained-release composition or a perfusion pump). Appropriate solidsupports can be, for example, calcium phosphate, magnesium stearate,magnesium carbonate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine and wax.

[0031] The pharmaceutical compositions containing a compound of theinvention can also be presented in liquid form such as, for example,solutions, emulsions, suspensions or a sustained-release formulation.Appropriate liquid supports can be, for example, water, organic solventssuch as glycerol or glycols such as polyethylene glycol, as well astheir mixtures, in varying proportions, in water.

[0032] The administration of a medicament according to the invention canbe carried out by topical, oral, parenteral route, by intramuscularinjection etc.

[0033] The dose of a compound according to the present invention, to beprovided for the treatment of the above-mentioned diseases or disorders,varies depending on the administration method, the age and body weightof the subject to be treated as well as the state of the latter, andwill be finally decided by the attending doctor or vet. Such a quantitydetermined by the attending doctor or vet is here called“therapeutically effective quantity”.

[0034] In accordance with the invention, the heterocarpine can beprepared by the process described hereafter.

Preparation of Heterocarpine

[0035] According to a preferred variant of the invention, in vitrocultures of calluses or of cell suspensions originating from differentorgans of the plant have been carried out. These tissues cultured onsemi-solid or liquid medium are able to bio-synthesize compounds havingbiological properties.

[0036] By “callus” is meant in the present Application a macroscopiccluster of undifferentiated cells of plants in culture on a semi-solidnutritive medium. The term “undifferentiated cells” in the presentApplication designates cells which have an aptitude under certainconditions to multiply in the form of a callus or of a cell suspensionwithout any morphogenesis phenomenon. Finally, by “cell suspension”, ismeant undifferentiated cells which can form microscopic clusters inculture in a liquid nutrition medium.

[0037] The choice of the nutritive medium, hormones, culture conditionsforms an integral part of the invention as well as the extraction andanalysis of the extracts from these in vitro cultures.

[0038] Cells from Pilocarpus Heterophyllus seeds can be cultured insuspension for example according to the procedure hereafter.

[0039] The organs are decontaminated according to the usual methodsbefore being cultured. Plantule organs in vitro have also served ascallogenesis starting material without requiring disinfectionbeforehand. The preferred basic nutritive medium is one of the mediacommonly used for in vitro culture: this is Gamborg's medium (describedin Gamborg et al., Nutrient requirements of suspension cultures ofSoybean root cells, Exp. Cell Res. (1968), 50(1), 151-158). The carbonsource is saccharose but glucose can also be used at a concentration of1 to 120 g/l, preferably approximately 30 g/l. The macro-elementscontent can also be reduced by a factor of 2. Auxin or an auxin and acytokinin are added to the medium, with a preference for a combinationof both hormones, generally 2,4-dichlorophenoxyacetic acid and kinetin,but α-xnaphthaleneacetic acid (NAA), β-indoleacetic acid (IAA),β-indolbutanoic acid (IBA) or picloram can also be combined with thekinetin or benzylaminopurine (BAP). The concentration can vary from 0.1to 10 mg/l for the auxin (for example 1 mg/l can be chosen), and from0.01 to 2 mg/l for the cytokinin (for example 0.06 mg/l can be chosen).The vitamins are those associated with the different basic media. Thecultures are carried out in light or in darkness. The temperature canvary from 10° C. to 33° C. but is preferentially approximately 23° C.The pH of the medium is comprised between 4 and 6.5 and ispreferentially adjusted to 5.8 before sterilization. Moreover, agar mayor may not be added to the medium.

[0040] The primary calluses appear after a few days of culture and canbe separated from the original implant, removed and subcultured afterapproximately 1 month then cultured on agar semi-solid medium (in tubesor Petri dishes), at intervals of 4 to 8 weeks, preferably 6 weeks, thusa callus can be kept for years by successive subcultures on new media.The callus can also be subcultured in a stirred liquid culture medium(Erlenmeyer flask or bioreactor) with subcultures at 2 to 6 weeks,preferably 3 weeks.

[0041] The strains obtained are distinguished by their genetic origin,culture conditions, appearance and absence of morphogenesis.

[0042] The lyophilized Pilocarpus Heterophylluscells are extracted withwater at a temperature of 0 to 50° C., and preferably 4 to 25° C. Theextract thus obtained is lyophilized before being redissolved at asuitable concentration (for example approximately 30% of dried matter).The proteins precipitated by the addition of a concentrated solution ofammonium sulphate (for example at a concentration representing 70 to 90%of the saturation concentration) are dissolved in a minimum of water andthe insoluble materials are recovered by centrifugation. The proteinsare then separated by column chromatography (the eluent preferably beingwater) and the heterocarpine (identifiable by its molecular mass ofapproximately 90.9 kDa) can then be recovered.

Preparation of Antibodies Specifically Binding Heterocarpine

[0043] The present invention provides binding agents, such as theantibodies which specifically bind heterocarpine. Such an agent isreferred to as “specifically binding” a protein if it reacts at adetectable level (for example by an ELISA test) with said protein: anddoes not detectably react with other proteins. “The binding” refers to anon-covalent association between 2 separate molecules such that acomplex is formed. The binding ability can be evaluated, for example, bydetermination of the binding constant for the formation of the complex.The binding constant is the value obtained when the value of the complexconcentration is divided by the product of the values of thenon-complexed component concentration. 2 products are called “bound”when the binding constant reaches 103 1/mol. The binding constant can bedetermined using methods well known to a person skilled in the art.

[0044] Any agent which can satisfy the above criteria can be regarded asa binding agent.

[0045] In the present invention, a binding agent is preferably anantibody or a fragment of the latter. The antibody can be prepared byany technique available to a person skilled in the art (cf. Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988). In general, the antibodies can be produced by cell culturetechniques including the generation of monoclonal antibodies or viatransfections of antibody genes into host cells from bacteria or mammalsin order to produce recombinant antibodies.

[0046] Among other techniques, use of those described hereafter ispreferred. An immunogen containing heterocarpine is injected into agroup of mammals (for example mice, rats, rabbits, sheep or goats). Inthis stage, the heterocarpine can serve as immunogen withoutmodification. Alternatively, a superior immune response can be inducedif the heterocarpine is combined with a transport protein such as bovineserum albumin or limpet haemocyanin. The immunogen is injected into thehost animal, preferably according to a predetermined schedule, and theanimals are bled periodically. Polyclonal antibodies specific toheterocarpine can thus be purified from such antiserum, for example, byaffinity chromatography using heterocarpine coupled with an appropriatesolid support.

Pharmaceutical Compositions Intended for the Release of GHRH

[0047] These compositions can in particular be prepared fromheterocarpine and GHRH according to one of the methods described in thejournal by De Wolf and Brett, Pharmacological Reviews (2000), 52,207-236 and the references cited therein.

[0048] Unless otherwise specified, all the technical and scientificterms used here have the same meaning as that usually understood by anordinary specialist in the field to which this invention belongs.Similarly, all the publications, patent applications, all the patentsand all other references mentioned here are incorporated by way ofreference.

[0049] The following examples are presented in order to illustrate theabove procedures and should in no event be considered as a limit to thescope of the invention.

OBTAINING THE HETEROCARPINE EXAMPLE 1

[0050] Culture of Cells in Vitro:

[0051] A Pilocarpus Heterophyllus seed is germinated and the stemresulting from this germination is removed. Said stem is cultured in aGamborg medium (Gamborg et al., Nutrient requirements of suspensioncultures of Soybean root cells, Exp. Cell Res. (1968), 50(1), 151-158)to which 30 g/l of saccharose, 1 mg/l of 2,4-dichlorophenoxyacetic acidand 0.06 mg/l of kinetin have been added. The culture is carried out intubes at a temperature of 23° C. and in darkness. Subcultures arecarried out every 6 weeks under usual conditions. The strains, which aregranular in appearance, have a beige pigmentation.

[0052] A growth kinetic of the strains, based on the increase in mass offresh and dried material from the biomass, was carried out over 8 weeks.The calluses from 2 tubes are combined and constitute a twice-weeklyharvest, the first harvest taking place at time 0. Calluses and geloseare then harvested and lyophilized. It is observed that growth isexponential up to 6 weeks of culture before the appearance of astationary growth phase.

[0053] Extraction of the Cell Cultures: 25 g of lyophilized PilocarpusHeterophyllus cells are extracted twice by immersion in 375 ml of waterat 4° C., and left overnight at 4° C., then in 250 ml of water at 4° C.for 4 hours and finally washed with 125 ml of water at 4° C. Eachaqueous solution thus obtained is filtered under vacuum through a glassfilter surmounted with celite in order to separate the cell debris fromthe aqueous solution. The aqueous solutions thus combined are thenlyophilized in order to obtain 9.4 g of dried material. The lyophilizeddry extract is then dissolved in 31 ml of water at 20° C. in order toobtain a solution containing 30% of dry extract. 17.4 g of ammoniumsulphate is added in small portions with constant magnetic stirring inorder to precipitate the protein fraction. The protein precipitate isthen separated from the ammonium sulphate solution by centrifugation at3000 rpm for 20 minutes. The ammonium sulphate solution is decanted andthe precipitated proteins are dissolved in 22 ml of water,re-centrifuged and filtered in order to eliminate the insolubleparticles.

[0054] The filtrate obtained is then subjected to gel-filtrationchromatography. It is injected into a column (Buchi N° 19678,L=230 mm;internal diameter=26 mm) filled with Superdex:™ 200 (Amersham PharmaciaBiotech, reference no. 17-1043-01; particles with an average diameter of13 μm) prepared according to the manufacturer's recommendations usingultra-pure water (Water's Milli-Q) as eluent at a flow rate of 5 ml perminute. 40 ml fractions are thus collected and the active protein isfound in the third and fourth fraction. These fractions are lyophilizedin order to obtain approximately 14.2 mg of active product.

[0055] The purity of the product obtained is demonstrated by theappearance of a single band on electrophoresis gel containing sodiumdodecylsulphate (SDS PAGE). The product corresponding to this band isdesignated hereafter as heterocarpine.

EXAMPLE 2

[0056] The cells cultured in vitro according to the same procedure asthat described in Example 1 above are extracted according to the methoddescribed hereafter. 100 g of lyophilized Pilocarpus Heterophyllus cellsare extracted using 2 litres of demineralized water at 20° C., themixture being maintained under stirring overnight. The cells and theextract are filtered by suction on frit (porosity 3, diameter 20 cm)covered by a celite bed (previously washed with acid; 1 to 2 cm thick).The cells recovered are washed with 400 ml of demineralized water beforebeing eliminated. The aqueous filtrate is then acidified to pH 3.0 bythe addition of approximately 10 ml of 18% hydrochloric acid. Theacidified solution is then delipidated by liquid-liquid extraction using400 ml of dichloromethane. The dichloromethane phase is decanted theneliminated. The delipidated solution is subjected to rotary evaporationin order to eliminate the residual dichloromethane. Approximately 30 gof polyvinylpyrrolidone is then added to the delipidated solution (pHapproximately 3.0) and the mixture is stirred for approximately 30minutes in order to eliminate the tannins. The mixture is filteredthrough a bed by suction on frit (porosity 3, diameter 10 cm) covered bya mixed bed comprising 25 g of celite (previously washed with acid) and25 g of polyvinylpyrrolidone. The filtrate is then passed through a bedof 400 ml of Diaion® HP-20 (Mitsubishi Chemical Company) pre-activatedaccording to the manufacturer's instructions. The resulting filtrate isthen rendered alkaline (pH 10) by the addition of approximately 60 ml ofa 20% ammonium hydroxide solution. A slight precipitation appears after30 minutes of rest. 1 g of celite (previously washed with acid) is addedto the alkaline solution which is then filtered by suction through amembrane filter (0.22 μm). Approximately 2 litres of filtrate are thenpassed through a HiPrep® Q XL 16/10 column, mounted on an Aktaopurificator and pre-balanced at pH 10.2 with a piperazine/HCl 0.1Mbuffer, with a flow rate of 0.5 ml per minute (the HiPrepe® column andthe Akta® purificator are both products from the company AmershamBiosciences). The column is then washed successively with 6 columnvolumes of the starting buffer at pH 10.2, 5 column volumes of the samebuffer containing a 0.2M concentration of NaCl; and 10 column volumes ofthe same buffer containing a 1M concentration of NaCl. The majority ofthe heterocarpine is recovered in the first 3 column volumes of buffercontaining the 1M concentration of NaCl. The active fractions aredesalinated by passing through a Sephadex® G25 column (volume of thebed: 260 ml) using demineralized water as eluent. The active fractions,found in the first column volume corresponding with the hold-up volume,are then lyophilized in order to obtain 170 mg of heterocarpine. Theheterocarpine thus obtained is practically single band on SDS PAGE gel.

CHARACTERIZATION OF THE HETEROCARPINE

[0057] Analysis and Micro-sequencing:

[0058] The samples are loaded onto a 10% polyacrylamide gel. Aftermigration, the gels are fixed and stained with Coomassie blue.

[0059] The gel tracks represented in FIG. 3 corresponding to tracks 1,2, 3, 4 and 5 are respectively the molecular weight marker (Amersham),0.5, 1 and 2 μg of the content of the final heterocarpine fraction asobtained in Example 1 and the molecular mass marker (Amersham).Determination of the molecular mass by means of a standard molecularmass marker graph using standard computing tools well known to a personskilled in the art (for example, Viber Lourmat's Bio-Profil Bio1Dsoftware) makes it possible to show that heterocarpine has a molecularmass of 90.9 kiloDaltons (±1.6 kiloDaltons).

[0060] For protein micro-sequencing analysis, the band of polyacrylamidecontaining the protein is cut out and digested in 300 μl of digestionbuffer containing 50 mM Tris (pH 8.6), 0.03% of sodium dodecylsulphateat 35° C. for 18 hours in the presence of 0.4 μg of endolysine-C(Sigma). The peptides obtained are separated, by HPLC, on a DEAE-C18in-line column 1 mm in diameter. The separation gradient is based on amixture of acetonitrile (from 2 to 70%) and 0.1% trifluoroacetic acid(TFA). The sequencing is then carried out on a Procise sequencer(Applied Biosystem). In this way three peaks have been sequenced, makingit possible to characterize heterocarpine in a unique manner. Thecorresponding sequences are identified in the present Application bySEQ.ID.NO. 1, SEQ.ID.NO. 2 and SEQ.ID.NO. 3.

[0061] Analysis of the glycoproteins is carried out by the detection ofsugared structures of the glycoproteins separated by SDS-PAGE gel. Thisdetection system is a modification of the “Periodic Acid-Schiff” methodsand leads to the appearance of magenta bands showing evidence of theglycoproteins (Sigma). For the heterocarpine as obtained in Example 1,the result reproduced in FIG. 4 is obtained.

PHARMACOLOGICAL PROPERTIES OF HETEROCARPINE

[0062] Stable Transfections of the Human GHRH Receptor (hGHRH-R):

[0063] Human embryo kidney cells, HEK-293, (a cell line developed by Dr.Stuart Sealfon, Mount Sinai Medical School, New York, N.Y.) expressingthe human GHRH receptor in stable manner were obtained from Dr. KellyMayo (Northwestern University, Chicago, Ill).

[0064] Cell Culture and Membrane Preparation:

[0065] The HEK-293 cells transfected in a stable manner with the humanGHRH receptor described above are cultured in DMEM (Dulbecco's modifiedEagle's medium, high glucose content; supplied by Life technologies)supplemented with 0.4 mg/ml of G418 (Life technologies) in the presenceof 10% of foetal calf serum and 4 mM of L-glutamine (Life technologies).The cells are homogenized in buffer A containing 50 mM HEPES (pH 7.4), 5mM of magnesium chloride (MgCl₂), 2 mM ofethyleneglycol-bis(2-amino-ethyl)-N,N,N′,N′-tetraa acid (EGTA) and 50μg/ml of bacitracin then are subjected to sonication in the same bufferA. The cells thus homogenized are centrifuged at 4° C. at 39,000 g for10 minutes, suspended in buffer A and re-centrifuged at 4° C. at 40,000g for 10 minutes. The total membrane proteins are quantified byBradford's technique. The pelleted membranes are thus stored at −80° C.for later use.

[0066] Competitive Binding Test on hGHRH-R:

[0067] The membranes of the HEK-293 cells transfected in a stable mannerwith the human GHRH receptor are diluted to a concentration of 100 μg/mlin the reaction buffer containing 50 mM HEPES (pH 7.4), 5 mM of MgCl₂, 2mM of EGTA, 50 μg/ml of bacitracin and 0.5% of bovine serum albumin(BSA). The membranes are incubated with 0.05 nM of [¹²⁵ I]GHRH(1-44amide) (Amersham) in a final volume of 200 μl in the presence ofincreasing concentrations of heterocarpine for 2 hours at 23° C. Thereaction is stopped by rapid filtration on 96-well GF/C filters 0.1%pre-loaded with polyethylenimine. The filters are then washed threetimes at 4° C. with washing buffer containing 50 mM Tris (pH 7.4) usinga Packard 96-well filtration station. The filters thus dried aresubmerged in 20 μl of scintillating cocktail (Microscint O, Packard) andare subjecting to Topcount counting (Packard). The non-specific activityis determined in the presence of 100 nM of hGHRH. A dose-response curveis generated for hGHRH (0.001 nM-100 riM) and the results obtained areincluded in FIG. 1. Competitive Formation of Cyclic AMP:

[0068] The HEK-293 cells transfected in a stable manner with the humanGHRH receptor are distributed in 48-well culture plates and cultured for3 days. The culture medium is then removed and replaced by medium Bcontaining 250 μl of DMEM (Dulbecco's modified Eagle's medium, highglucose content; supplied by Life technologies) in the presence of 0.5%of BSA, 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX) and pre-incubatedfor 5 minutes at 37° C. At the end of the pre-incubation period, theheterocarpine is tested for an additional 20 minutes. The concentrationsobserved are reported in FIG. 2. The incubation is stopped by theaddition of 100 μl of O.1M HCl and the aliquots are analysed for theircyclic AMP content using the FlashPlate kit (New England Nuclear).

[0069] Assay of GH in Rats:

[0070] Levels of GH in rats (Sprague Dawley males) are measured in bloodsamples by an enzymo-immunological test developed by Spi-Bio (Spi-Bio,France). The rats are treated by intravenous injection of heterocarpineat increasing doses (vehicle alone, 1, 3 and 10 nmol), then, 10 minutesafter, by intravenous injection of 10 μg (3 nmol) of hGHRH. Ten minutesafter injection of the hGHRH, the growth hormone levels are measured inthe blood samples as described above. The results obtained arerepresented in FIG. 5.

BRIEF DESCRIPTION OF THE FIGURES

[0071]FIG. 1 is a graph representing the inhibition of binding of humanGHRH on the human GHRH receptor as a function of increasingconcentrations of heterocarpine.

[0072]FIG. 2 is a graph representing the inhibition of the production ofcyclic AMP in cells transfected in a stable manner with the human GHRHreceptor in the presence of 10 nM of human GHRH as a function ofincreasing concentrations of heterocarpine.

[0073]FIG. 3 is a reproduction of an SDS-PAGE protein gel plate showingthe presence of heterocarpine having a molecular weight of 90.9 kDa.

[0074]FIG. 4 is a reproduction of an SDS-PAGE protein gel plate showingthat heterocarpine is a glycoprotein (Panel B).

[0075]FIG. 5 is a representation in histogram form representing theinhibition of GH synthesis in rats in the presence of 10 μg of humanGHRH as a function of increasing concentrations of heterocarpine.

1 3 1 10 PRT Pilocarpus Heterophyllus 1 Lys Leu Ile Gly Ala Arg Tyr PheAsp Lys 1 5 10 2 14 PRT Pilocarpus Heterophyllus 2 Tyr Gly Glu Asp IleIle Val Gly Val Ile Asp Ser Gly Val 1 5 10 3 6 PRT PilocarpusHeterophyllus 3 Pro Glu Ser Glu Ser Tyr 1 5

1. Isolated protein which can be obtained by extraction from the plantPilocarpus heterophyllus, which is characterized in that it has amolecular mass of approximately 90.9 kDa and comprises fragments ofpeptide sequences SEQ.ID.NO. 1, SEQ.ID.NO. 2 and SEQ.ID.NO. 3; saidprotein be able to be presented in a glycosylated or nonglycosylatedform.
 2. Protein according to claim 1, characterized in that it has beenobtained from an extract from cells of the plant PilocarpusHeterophyllus cultured in vitro.
 3. As a medicament, a protein accordingto claim 1 or
 2. 4. Monoclonal antibody, or an antigen binding fragmentof the latter, which specifically binds the isolated protein of claim 1.5. As a medicament, a monoclonal antibody, or an antigen bindingfragment of the latter, which specifically binds the isolated protein ofclaim
 1. 6. Pharmaceutical composition comprising, as active ingredient,a protein according to claim 1 or 2 , as well as one or morepharmaceutically acceptable excipients.
 7. Use of a protein according toclaim 1 or 2 for preparing a medicament intended to antagonize theeffects of GHRH.
 8. Use of a protein according to claim 1 or 2 forpreparing a medicament intended to treat a proliferative disease, inparticular cancer.
 9. Use of a protein according to claim 1 or 2 forpreparing a medicament intended to treat acromegaly.
 10. Use of aprotein according to claim 1 or 2 for preparing a medicament intended totreat diabetic retinopathies and nephropathies.
 11. Process ofextraction and isolation of heterocarpine from cells of the plantPilocarpus Heterophyllus comprising a stage of extraction of the cellsof the plant Pilocarpus Heterophyllus with water at a temperature of 0to 50 ° C., said extraction stage being followed by a filtration stageto separate the heterocarpine-rich filtrate from the cells of PilocarpusHeterophyllus and by one or more separation stages of the heterocarpinefrom the other components extracted from the plant PilocarpusHeterophyllus.